The proposed research project is concerned with defining the sequence of events surrounding the in vitro synthesis of proteins and their subsequent transfer into intact chloroplasts. Based on preliminary results we have postulated that proteins which are destined for the chloroplast are synthesized on free cytoplasmic polysomes as larger precursors which are transported into the organelle only after the entire molecule had been synthesized. The chain extension in these precursors contains the sequence information necessary for the specific transport. Experiments will be carried out with spinach in order to test this hypothesis. Three representative chloroplast proteins (the small subunit of Ru-1,5-P2 carboxylase, the chlorophyll-binding protein of CP II, and ferredoxin) which have different properties and locations inside the organelle will be investigated. Polyadenylated RNA and free cytoplasmic polysomes will be obtained from spinach and translated in the wheat germ or reticulocyte lysate system. Radioactive precursors will be isolated by sequential immunoabsorption using monospecific antibodies raised against each of the three proteins. In vitro reconstitution experiments will be carried out to show that the precursors but not the authentic proteins are transported into intact chloroplasts. The endoprotease(s) which converts the precursor into authentic protein will be localized and characterized. Radiosequencing experiments will be carried out to determine the partial or complete amino acid sequence in the chain extension and to establish that the precursors have been processed correctly in vitro.